Biomedical Light Microscopy

1. Light microscopy as an optical system, the stand and its parts.- 1.1 Basic theory.- 1.2 The objective as an optical tool; resolving power.- 1.3 Eyepieces.- 1.4 Objective and eyepiece as an integrated system.- 1.4.1 The interplay between objective and eyepiece.- 1.4.2 Tube length.- 1.4.3 Axial resolving power and depth of field.- Recommended further reading.- 2. The light microscope as a tool for observation and measurement: illumination and image formation.- 2.1 Modulation of the illuminating light by the object.- 2.2 The stand and its parts.- 2.3 Illumination and image formation.- 2.3.1 General aspects.- 2.3.2 Types of illumination.- 2.3.3 Special types of illumination: darkground illumination.- 2.3.4 The light source.- 2.3.5 Confocal illumination.- Recommended further reading.- 3. Fluorescence microscopy.- 3.1 Theoretical background.- 3.1.1 What is fluorescence?.- 3.1.2 Physical properties of fluorescence.- 3.1.3 Spectral properties of fluorochromes.- 3.1.4 Quantum efficiency of fluorochromes.- 3.2 The fluorescence microscope.- 3.2.1 Incident or transmitted light illumination.- 3.2.2 Fluorescence microscopy with transmitted illumination.- 3.2.3 Fluorescence microscopy with incident illumination.- 3.2.4 Components of the fluorescence microscope.- 3.2.5 The two-wavelengths excitation method for fluorescence microscopy with incident light.- Recommended further reading.- 4. Special optical techniques of image formation.- 4.1 Phase-contrast microscopy.- 4.1.1 Basal theoretical facts.- 4.1.2 Practical realization of the phase-contrast.- 4.1.3 The phase-contrast image with different objects.- 4.2 Interferometry and interference contrast.- 4.2.1 Principles of image formation in interference contrast.- 4.2.2 Differential interference contrast.- 4.3 Modulation-contrast microscopy.- 4.4 Polarization microscopy.- 4.4.1 Anisotropy as an optical phenomenon.- 4.4.2 The polarized light microscope.- 4.5 Reflection microscopy and reflection-contrast microscopy.- 4.6 Acoustic microscopy.- 4.7 Superresolution: modern developments.- Recommended further reading.- 5. Reproduction of microscopic images, microphotography.- 5.1 Drawing and drawing apparatuses.- 5.2 Microprojection.- 5.3 Television microscopy.- 5.4 Photomicrography.- 5.4.1 Some basic principles.- 5.4.2 Photographic materials.- 5.4.3 Photomicrography in practice.- 5.4.4 Colour photomicrography.- 5.4.5 Photomicrography of fluorescence images.- 5.4.6 Special techniques in microphotography.- 5.4.7 Holographic photomicroscopy.- 5.4.8 Cinemicrography.- Recommended further reading.- 6. Quantitative analysis of microscopic images.- 6.1 Introduction.- 6.2 Morphometric techniques.- 6.2.1 Estimation of distances perpendicular to the optical axis.- 6.2.2 Measurements of distances along the optical axis.- 6.2.3 Measurements of surfaces and volumes: stereology.- 6.3 Counting methods.- 6.4 Absorption and fluorescence measurement of cells.- 6.5 Absorption cytophotometry (cytophotometry or microphotometry).- 6.5.1 Object plane scanners.- 6.5.2 Image plane scanners.- 6.6 Fluorescence cytophotometry (cytofluorometry, microfluorometry).- 6.6.1 Theoretical background.- 6.6.2 Practical aspects of cytofluorometry.- 6.7 Flow cytometry.- 6.8 Microspectrophotometry.- Recommended further reading.- 7. Automation: image analysis and pattern recognition.- 7.1 General introduction.- 7.2 Scanning of microscopic objects: special cameras.- 7.3 The digitized image.- 7.3.1 Image processing and image analysis.- 7.3.2 Spatial resolution and grey value resolution.- 7.3.3 Intensity transformations.- 7.3.4 Segmentation of images.- 7.4 Image analysis.- 7.5 Pattern recognition.- Recommended further reading.- 8. Appendix: technical aspects of the microscopical observation in practice.- 8.1 Introduction.- 8.2 Setting up a microscope for Kohler illumination.- 8.3 Again: the object.- 8.4 On the way through the object.- 8.5 Maintenance and minor technical problems.- 8.6 Frequently occurring minor defects.- Recommended further reading.- Index of subjects.